Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals

We conducted experiments to determine whether lambs fed grain prefer foods and solutions containing sodium bicarbonate (NaHCO3) and lasalocid, compounds capable of attenuating acidosis. In Exp. 1, we determined whether lambs fed barley preferred flavored rabbit pellets (RP) containing NaHCO3 and lasalocid. Lambs in two groups (n = 10/group) were fed increasing amounts of barley on d 1 to 12 (300 to 1,100 g) and again on d 23 to 34 (300 to 1,350 g). After ingesting barley on d 1 to 12, lambs were fed ground RP containing lasalocid and NaHCO3 (i.e., medicated) and flavored with either 2% onion (group 1) or 2% oregano (group 2). During d 23 to 34, lambs were fed unmedicated RP containing NaCl and flavored with either 2% oregano (group 1) or 2% onion (group 2). During preference tests on d 35 to 40, lambs fed grain preferred RP with NaHCO3 to RP with NaCl (151 vs. 96 g; P < .01). In the Exp. 2, we determined whether wheat ingestion affected consumption of aqueous solutions containing NaHCO3. In trial 1, 28 lambs were assigned to four treatments: 1) low-wheat + 2% NaHCO3, 2) high-wheat + 2% NaHCO3, 3) low-wheat + water, and 4) high-wheat + water. For 12 d from 0800 to 0830, lambs in treatments 1 and 3 were fed 300 g of wheat and lambs in treatments 2 and 4 were fed up to 1,300 g of wheat; fluids (NaHCO3 and water) were then offered from 0930 to 1230 daily. Lambs drank more NaHCO3 on the high- than on the low-wheat diet (1,332 vs 890 g; P = .03); water consumption was similar for lambs on the high- and low-wheat diets (1,675 vs 1,700 g; P > .10). In trial 2, lambs in treatments 3 and 4 were offered a solution containing 1.4% NaCl. For 13 d from 0800 to 0830, lambs in treatments 1 and 3 were fed 500 g of wheat and lambs in treatments 2 and 4 were fed up to 1,700 g of wheat. Lambs had access to fluids from 0800 to 1200 daily. Lambs drank nearly twice as much NaHCO3 solution on the high- than on the low-wheat diet (1,066 vs 572 g), whereas they drank only 1.4 times more NaCl solution on the high- than on the low-wheat diet (888 vs. 634 g; P < .001). Fewer lambs showed signs of acidosis in treatment 2 than in treatment 4 in trials 1 (2 vs 9) and 2 (7 vs 17). Collectively, these results are consistent with the hypothesis that lambs fed grain prefer substances that attenuate acidosis.     

Single amino acid substitution in constant region 1 or 4 of gp120 causes the phenotype of a human immunodeficiency virus type 1 variant with mutations in hypervariable regions 1 and 2 to revert

The second major cysteine loop of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains 5 to 11 consensus N-linked glycosylation sites, which is disproportionately higher than the number of such sites found in other regions of gp120. Amino acid substitutions introduced at three of six N-linked glycosylation sites in this region of an infectious molecular clone, HXB2, resulted in severe impairment of virus infectivity. Isolation and genetic characterization of a revertant of this mutant revealed an isoleucine-for-valine substitution at position 84 in constant region 1 and an isoleucine-for-methionine substitution at position 434 in constant region 4. Further mutational analysis indicated that either isoleucine substitution was sufficient to confer the revertant phenotype. These findings demonstrate that V1/V2 not only functionally interacts with C4, as previously reported, but also interacts with C1. The observation that compensatory changes do not involve regeneration of N-linked glycosylation sites in the second major cysteine loop suggests that replication of human immunodeficiency virus type 1 in vitro is independent of the presence of a disproportionate number of N-linked glycosylation sites within this loop.     

Specific substitutions at amino acid 256 of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase mediate resistance to thapsigargin in thapsigargin-resistant hamster cells

High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.     

Twenty-four-hour L-[1-(13)C]tyrosine and L-[3,3-(2)H2]phenylalanine oral tracer studies at generous, intermediate, and low phenylalanine intakes to estimate aromatic amino acid requirements in adults

Daily pattern and rates of whole-body tyrosine oxidation and phenylalanine hydroxylation were determined in young adults (15 men, 1 woman) receiving [13C]tyrosine and [(2)H2]phenylalanine via primed, constant oral infusion and [(2)H4]tyrosine by vein (five subjects also received [(2)H3]leucine simultaneously by vein) continuously for 24 h (12 h fast then 12 h fed). Subjects were given a diet supplying 96.6 (n = 5), 35.6 (the proposed requirement; n = 5), and 18.5 mg phenylalanine x kg(-1) x d(-1) (n = 6) based on an otherwise adequate L-amino acid mixture for 6 d before the 24-h tracer study began. [Each diet was low in tyrosine: 6.79 mg x kg(-1) x d(-1).] Our hypothesis was that subjects would be in tyrosine equilibrium, positive balance, or both, at the 96.6- and 35.6-mg intakes and in distinctly negative balance at the 18.5-mg intake. The diurnal pattern in phenylalanine and tyrosine kinetics was dependent on the intake and, presumably, on the adequacy of dietary phenylalanine. Wholebody tyrosine balances, determined from rates of phenylalanine hydroxylation and tyrosine input and oxidation were negative (0.05 < P < 0.1 from zero balance) with the low (18.5 mg) phenylalanine intake [total aromatic amino acid (AAA) intake: 25.3 mg x kg(-1) x d(-1)] but at equilibrium (P > 0.05 from zero balance) with the two higher phenylalanine intakes. Whole-body AAA balance (AAA intake - tyrosine oxidation) was negative (P < 0.05 from zero balance) with the low intake, at equilibrium with the intermediate intake, and apparently distinctly positive (P < 0.05) with the generous intake. Despite model limitations, as discussed, these findings lend further support for a proposed, tentative value for a total mean requirement of 39 mg AAA x kg(-1) x d(-1).